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Proximity Extension Assay (PEA) and mass spectrometry (MS) methodologies were utilized for the proteomic and lipidomic characterization of plasma specimens from patients who developed Alzheimer's disease. Proteomics was performed by both PEA and Liquid Chromatography (LC)/MS in this study, but all the more because LC/MS generally tends to be biased towards proteins with high expression and high variability, generating hypotheses proved challenging. Consequently, attempt was made to interpret the results from the PEA data. There were 150 significantly variable proteins and 68 lipids among 1000 proteins and 400 lipids. Pathway analysis was performed for both total and variable proteins measured to reduce bias, and it appeared that vascular fragility was related to AD. Furthermore, a multitude of lipid-associated proteins exhibited statistical changes. In certain instances, the function of individual proteins affected the factors associated with them, whereas others demonstrated trends contrary to anticipated outcomes. These trends seem indicative of diverse feedback mechanisms that provide homeostatic equilibrium. The degree of unsaturation of fatty acids, correlated with cardiovascular risk, warrants specific attention. Certain bile acids exhibited the potential to cause vascular endothelial damage. Contemplating these discoveries in tandem with previously documented phenomena, subtle shifts in homeostatic functions seem to be linked to the fragility of vascular endothelial cells. This is evidenced by the slow and chronic evolution of Alzheimer's disease from preclinical stages to its manifestation.
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Enfermedad de Alzheimer , Lipidómica , Proteómica , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/sangre , Humanos , Lipidómica/métodos , Proteómica/métodos , Masculino , Femenino , Anciano , Cromatografía Liquida , Lípidos/sangre , Anciano de 80 o más Años , Espectrometría de Masas/métodosRESUMEN
Although the herpes simplex virus type 1 (HSV-1) genome was thought to contain approximately 80 different protein coding sequences (CDSs), recent multi-omics analyses reported HSV-1 encodes more than 200 potential CDSs. However, few of the newly identified CDSs were confirmed to be expressed at the peptide or protein level in HSV-1-infected cells. Furthermore, the impact of the proteins they encode on HSV-1 infection is largely unknown. This study focused on a newly identified CDS, UL31.6. Re-analyzation of our previous chemical proteomics data verified that UL31.6 was expressed at the peptide level in HSV-1-infected cells. Antisera raised against a viral protein encoded by UL31.6 (pUL31.6) reacted with a protein with an approximate molecular mass of 37 kDa in lysates of Vero cells infected with each of three HSV-1 strains. pUL31.6 was efficiently dissociated from virions in high-salt solution. A UL31.6-null mutation had a minimal effect on HSV-1 gene expression, replication, cell-to-cell spread, and morphogenesis in Vero cells; in contrast, it significantly reduced HSV-1 cell-to-cell spread in three neural cells but not in four non-neural cells including Vero cells. The UL31.6-null mutation also significantly reduced the mortality and viral replication in the brains of mice after intracranial infection, but had minimal effects on pathogenic manifestations in and around the eyes, and viral replication detected in the tear films of mice after ocular infection. These results indicated that pUL31.6 was a tegument protein and specifically acted as a neurovirulence factor by potentially promoting viral transmission between neuronal cells in the central nervous system.IMPORTANCERecent multi-omics analyses reported the herpes simplex virus type 1 (HSV-1) genome encodes an additional number of potential coding sequences (CDSs). However, the expressions of these CDSs at the peptide or protein levels and the biological effects of these CDSs on HSV-1 infection remain largely unknown. This study annotated a cryptic orphan CDS, termed UL31.6, an HSV-1 gene that encodes a tegument protein with an approximate molecular mass of 37 kDa, which specifically acts as a neurovirulence factor. Our study indicates that HSV-1 proteins important for viral pathogenesis remain to be identified and a comprehensive understanding of the pathogenesis of HSV-1 will require not only the identification of cryptic orphan CDSs using emerging technologies but also step-by-step and in-depth analyses of each of the cryptic orphan CDSs.
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Herpesvirus Humano 1 , Proteínas Virales , Replicación Viral , Animales , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/patogenicidad , Herpesvirus Humano 1/fisiología , Chlorocebus aethiops , Células Vero , Ratones , Proteínas Virales/genética , Proteínas Virales/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Herpes Simple/virología , Virulencia , Femenino , HumanosRESUMEN
In recent years, research has explored the use of microRNA (miRNA) analysis in extracellular vesicles (EVs) as a minimally invasive strategy for the diagnosis and prediction of diseases. This is because miRNAs in EVs partly reflect the miRNA information and cellular status of the origin cells. However, not all intracellular miRNAs are internalized into EVs. Therefore, the miRNA information obtained from EVs is limited. To get more miRNA information, we aimed to produce artificial EVs (aEVs) encapsulating Argonaute 2 (Ago2) miRNA-binding protein, which actively incorporate miRNAs within themselves. In this study, we utilized the protein EPN-01, which is capable of releasing aEVs encapsulating it and associated proteins. This system enables us to obtain more miRNA species and increase each miRNA's yield in the EV fraction. Furthermore, we examined whether miRNAs in the EV fraction using our system reflect the cellular condition. In cells treated with CoCl2, a reagent for inducing a hypoxia-mimic state, we detected a change in the level of hypoxia marker miR-210 with aEVs. To the best of our knowledge, this is the first report on a method to increase the yield and variety of endogenous miRNAs in the EV fraction. This approach leads to improved accuracy of cell status assessment using miRNAs in EVs.
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Vesículas Extracelulares , MicroARNs , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Hipoxia/metabolismoRESUMEN
Many membraneless organelles (MLOs) formed through phase separation play crucial roles in various cellular processes. Although these MLOs co-exist in cells, how they maintain their independence without coalescence or engulfment remains largely unknown. Here, we investigated the molecular mechanism by which paraspeckles with core-shell architecture scaffolded by NEAT1_2 long noncoding RNAs exist as distinct MLOs. We identified NEAT1 deletion mutants that assemble paraspeckles that are incorporated into nuclear speckles. Several paraspeckle proteins, including SFPQ, HNRNPF and BRG1, prevent this incorporation and thus contribute to the segregation of paraspeckles from nuclear speckles. Shell localization of these proteins in the paraspeckles, which is determined by NEAT1_2 long noncoding RNA domains, is required for this segregation process. Conversely, U2-related spliceosomal proteins are involved in internalizing the paraspeckles into nuclear speckles. This study shows that the paraspeckle shell composition dictates the independence of MLOs in the nucleus, providing insights into the importance of the shell in defining features and functions of MLOs.
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Núcleo Celular , ARN Largo no Codificante , Condensados Biomoleculares , Núcleo Celular/genética , Núcleo Celular/metabolismo , Paraspeckles , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , HumanosRESUMEN
Mbtd1 (mbt domain containing 1) encodes a nuclear protein containing a zinc finger domain and four malignant brain tumor (MBT) repeats. We previously generated Mbtd1-deficient mice and found that MBTD1 is highly expressed in fetal hematopoietic stem cells (HSCs) and sustains the number and function of fetal HSCs. However, since Mbtd1-deficient mice die soon after birth possibly due to skeletal abnormalities, its role in adult hematopoiesis remains unclear. To address this issue, we generated Mbtd1 conditional knockout mice and analyzed adult hematopoietic tissues deficient in Mbtd1. We observed that the numbers of HSCs and progenitors increased and Mbtd1-deficient HSCs exhibited hyperactive cell cycle, resulting in a defective response to exogenous stresses. Mechanistically, we found that MBTD1 directly binds to the promoter region of FoxO3a, encoding a forkhead protein essential for HSC quiescence, and interacts with components of TIP60 chromatin remodeling complex and other proteins involved in HSC and other stem cell functions. Restoration of FOXO3a activity in Mbtd1-deficient HSCs in vivo rescued cell cycle and pool size abnormalities. These findings indicate that MBTD1 is a critical regulator for HSC pool size and function, mainly through the maintenance of cell cycle quiescence by FOXO3a.
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Médula Ósea , Células Madre Hematopoyéticas , Animales , Ratones , Ciclo Celular/genética , Hematopoyesis/genética , Células Madre Hematopoyéticas/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Factores de Transcripción/metabolismoRESUMEN
Wastewater-based epidemiology (WBE) is a promising tool to efficiently monitor COVID-19 prevalence in a community. For WBE community surveillance, automation of the viral RNA detection process is ideal. In the present study, we achieved near full-automation of a previously established method, COPMAN (COagulation and Proteolysis method using MAgnetic beads for detection of Nucleic acids in wastewater), which was then applied to detect SARS-CoV-2 in wastewater for half a year. The automation line employed the Maholo LabDroid and an automated-pipetting device to achieve a high-throughput sample-processing capability of 576 samples per week. SARS-CoV-2 RNA was quantified with the automated COPMAN using samples collected from two wastewater treatment plants in the Sagami River basin in Japan between 1 November 2021 and 24 May 2022, when the numbers of daily reported COVID-19 cases ranged from 0 to 130.3 per 100,000 inhabitants. The automated COPMAN detected SARS-CoV-2 RNA from 81 out of 132 samples at concentrations of up to 2.8 × 105 copies/L. These concentrations showed direct correlations with subsequently reported clinical cases (5-13 days later), as determined by Pearson's and Spearman's cross-correlation analyses. To compare the results, we also conducted testing with the EPISENS-S (Efficient and Practical virus Identification System with ENhanced Sensitivity for Solids, Ando et al., 2022), a previously reported detection method. SARS-CoV-2 RNA detected with EPISENS-S correlated with clinical cases only when using Spearman's method. Our automated COPMAN was shown to be an efficient method for timely and large-scale monitoring of viral RNA, making WBE more feasible for community surveillance.
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COVID-19 , ARN Viral , Humanos , Aguas Residuales , SARS-CoV-2/genética , COVID-19/diagnóstico , AutomatizaciónRESUMEN
Protein modification by ubiquitin-like proteins (UBLs) amplifies limited genome information and regulates diverse cellular processes, including translation, autophagy and antiviral pathways. Ubiquitin-fold modifier 1 (UFM1) is a UBL covalently conjugated with intracellular proteins through ufmylation, a reaction analogous to ubiquitylation. Ufmylation is involved in processes such as endoplasmic reticulum (ER)-associated protein degradation, ribosome-associated protein quality control at the ER and ER-phagy. However, it remains unclear how ufmylation regulates such distinct ER-related functions. Here we identify a UFM1 substrate, NADH-cytochrome b5 reductase 3 (CYB5R3), that localizes on the ER membrane. Ufmylation of CYB5R3 depends on the E3 components UFL1 and UFBP1 on the ER, and converts CYB5R3 into its inactive form. Ufmylated CYB5R3 is recognized by UFBP1 through the UFM1-interacting motif, which plays an important role in the further uyfmylation of CYB5R3. Ufmylated CYB5R3 is degraded in lysosomes, which depends on the autophagy-related protein Atg7- and the autophagy-adaptor protein CDK5RAP3. Mutations of CYB5R3 and genes involved in the UFM1 system cause hereditary developmental disorders, and ufmylation-defective Cyb5r3 knock-in mice exhibit microcephaly. Our results indicate that CYB5R3 ufmylation induces ER-phagy, which is indispensable for brain development.
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Autofagia , Citocromo-B(5) Reductasa , Retículo Endoplásmico , Ubiquitinas , Animales , Ratones , Autofagia/fisiología , Proteínas de Ciclo Celular/metabolismo , Citocromo-B(5) Reductasa/química , Citocromo-B(5) Reductasa/metabolismo , Retículo Endoplásmico/metabolismo , Procesamiento Proteico-Postraduccional , Ubiquitinación/fisiología , Ubiquitinas/química , Ubiquitinas/metabolismoRESUMEN
Cells can respond to stalled ribosomes by sensing ribosome collisions and employing quality control pathways. How ribosome stalling is resolved without collisions, however, has remained elusive. Here, focusing on noncolliding stalling exhibited by decoding-defective ribosomes, we identified Fap1 as a stalling sensor triggering 18S nonfunctional rRNA decay via polyubiquitination of uS3. Ribosome profiling revealed an enrichment of Fap1 at the translation initiation site but also an association with elongating individual ribosomes. Cryo-EM structures of Fap1-bound ribosomes elucidated Fap1 probing the mRNA simultaneously at both the entry and exit channels suggesting an mRNA stasis sensing activity, and Fap1 sterically hinders the formation of canonical collided di-ribosomes. Our findings indicate that individual stalled ribosomes are the potential signal for ribosome dysfunction, leading to accelerated turnover of the ribosome itself.
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Biosíntesis de Proteínas , Ribosomas , Estabilidad del ARN , ARN Mensajero/metabolismo , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Ribosomas/metabolismoRESUMEN
Induced differentiation is one of the most experience- and skill-dependent experimental processes in regenerative medicine, and establishing optimal conditions often takes years. We developed a robotic AI system with a batch Bayesian optimization algorithm that autonomously induces the differentiation of induced pluripotent stem cell-derived retinal pigment epithelial (iPSC-RPE) cells. From 200 million possible parameter combinations, the system performed cell culture in 143 different conditions in 111 days, resulting in 88% better iPSC-RPE production than that obtained by the pre-optimized culture in terms of the pigmentation scores. Our work demonstrates that the use of autonomous robotic AI systems drastically accelerates systematic and unbiased exploration of experimental search space, suggesting immense use in medicine and research.
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Células Madre Pluripotentes Inducidas , Procedimientos Quirúrgicos Robotizados , Teorema de Bayes , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Medicina Regenerativa , Epitelio Pigmentado de la RetinaRESUMEN
Influenza viral particles are assembled at the plasma membrane concomitantly with Rab11a-mediated endocytic transport of viral ribonucleoprotein complexes (vRNPs). The mechanism of spatiotemporal regulation of viral budozone formation and its regulatory molecules on the endocytic vesicles remain unclear. Here, we performed a proximity-based proteomics approach for Rab11a and found that ARHGAP1, a Rho GTPase-activating protein, is transported through the Rab11a-mediated apical transport of vRNP. ARHGAP1 stabilized actin filaments in infected cells for the lateral clustering of hemagglutinin (HA) molecules, a viral surface membrane protein, to the budozone. Disruption of the HA clustering results in the production of virions with low HA content, and such virions were less resistant to protease and had enhanced antigenicity, presumably because reduced clustering of viral membrane proteins exposes hidden surfaces. Collectively, these results demonstrate that Rab11a-mediated endocytic transport of ARHGAP1 with vRNPs stimulates budozone formation to ensure the integrity of virion surface required for viral survival. IMPORTANCE The endocytic transport of the influenza viral genome triggers the clustering of viral membrane proteins at the plasma membrane to form the viral budozone. However, host factors that promote viral budozone formation in concert with viral genome transport have not been identified. Here, we found that ARHGAP1, a negative regulator of the Rho family protein, is transported with the viral genome and stabilizes actin filaments to promote budozone formation. We have shown that ARHGAP1-mediated efficient formation of viral budozone was crucial for the clustering of viral HA protein to the progeny viral particles. The clustering of HA proteins on the virions is responsible for the structural integrity of the viral particles, which promotes viral stability and viral immune evasion. This study highlights the molecular mechanism that works in concert with viral genome packaging to ensure the structural integrity of viral particles.
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Gripe Humana , Proteínas Activadoras de GTPasa/genética , Genoma Viral , Humanos , Proteínas de la Matriz Viral/metabolismo , Proteínas Virales/metabolismo , Virión/genética , Virión/metabolismo , Ensamble de Virus/fisiologíaRESUMEN
This study developed a system consisting of two rounds of screening cellular proteins involved in the nuclear egress of herpes simplex virus 1 (HSV-1). Using this system, we first screened cellular proteins that interacted with the HSV-1 nuclear egress complex (NEC) consisting of UL34 and UL31 in HSV-1-infected cells, which are critical for the nuclear egress of HSV-1, by tandem affinity purification coupled with mass spectrometry-based proteomics technology. Next, we performed CRISPR/Cas9-based screening of live HSV-1-infected reporter cells under fluorescence microscopy using single guide RNAs targeting the cellular proteins identified in the first proteomic screening to detect the mislocalization of the lamin-associated protein emerin, which is a phenotype for defects in HSV-1 nuclear egress. This study focused on a cellular orphan transporter SLC35E1, one of the cellular proteins identified by the screening system. Knockout of SLC35E1 reduced HSV-1 replication and induced membranous invaginations containing perinuclear enveloped virions (PEVs) adjacent to the nuclear membrane (NM), aberrant accumulation of PEVs in the perinuclear space between the inner and outer NMs and the invagination structures, and mislocalization of the NEC. These effects were similar to those of previously reported mutation(s) in HSV-1 proteins and depletion of cellular proteins that are important for HSV-1 de-envelopment, one of the steps required for HSV-1 nuclear egress. Our newly established screening system enabled us to identify a novel cellular protein required for efficient HSV-1 de-envelopment. IMPORTANCE The identification of cellular protein(s) that interact with viral effector proteins and function in important viral procedures is necessary for enhancing our understanding of the mechanics of various viral processes. In this study, we established a new system consisting of interactome screening for the herpes simplex virus 1 (HSV-1) nuclear egress complex (NEC), followed by loss-of-function screening to target the identified putative NEC-interacting cellular proteins to detect a defect in HSV-1 nuclear egress. This newly established system identified SLC35E1, an orphan transporter, as a novel cellular protein required for efficient HSV-1 de-envelopment, providing an insight into the mechanisms involved in this viral procedure.
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Herpesvirus Humano 1 , Proteínas de Transporte de Membrana , Liberación del Virus , Animales , Sistemas CRISPR-Cas , Chlorocebus aethiops , Técnicas de Inactivación de Genes , Células HEK293 , Células HeLa , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiología , Humanos , Proteínas de Transporte de Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares , Proteómica , Células Vero , Proteínas Virales/metabolismoRESUMEN
It has not been considered that nephrons regenerate in adult mammals. We present that an organ-derived extracellular matrix in situ induces nephron regeneration in a preclinical model. A porcine kidney-derived extracellular matrix was sutured onto the surface of partial nephrectomy (PN)-treated kidney. Twenty-eight days after implantation, glomeruli, vessels, and renal tubules, characteristic of nephrons, were histologically observed within the matrix. No fibrillogenesis was observed in the matrix nor the matrix-sutured kidney, although this occurred in a PN kidney without the matrix, indicating the structures were newly induced by the matrix. The expression of renal progenitor markers, including Sall1, Six2, and WT-1, within the matrix supported the induction of nephron regeneration by the matrix. Furthermore, active blood flow was observed inside the matrix using computed tomography. The matrix provides structural and functional foundations for the development of cell-free scaffolds with a remarkably low risk of immune rejection and cancerization.
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Unlimited organ availability would represent a paradigm shift in transplantation. Long-term in vivo engraftment and function of scaled-up bioengineered liver grafts have not been previously reported. In this study, we describe a human-scale transplantable liver graft engineered on a porcine liver-derived scaffold. We repopulated the scaffold parenchyma with primary hepatocytes and the vascular system with endothelial cells. For in vivo functional testing, we performed auxiliary transplantation of the repopulated scaffold in pigs with induced liver failure. It was observed that the auxiliary bioengineered liver graft improved liver function for 28 days and exhibited upregulation of liver-specific genes. This study is the first of its kind to present 28 days of posttransplant evaluation of a bioengineered liver graft using a preclinical large animal model. Furthermore, it provides definitive evidence for the feasibility of engineering human-scale transplantable liver grafts for clinical applications.
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Fallo Hepático , Trasplante de Hígado , Animales , Células Endoteliales , Hepatocitos/trasplante , Hígado/irrigación sanguínea , Porcinos , Ingeniería de Tejidos , Andamios del TejidoRESUMEN
Recent studies have shown that adipose tissue is an immunological organ. While inflammation in energy-storing white adipose tissues has been the focus of intense research, the regulatory mechanisms of inflammation in heat-producing brown adipose tissues remain largely unknown. We previously identified apoptosis signal-regulating kinase 1 (ASK1) as a critical regulator of brown adipocyte maturation; the PKA-ASK1-p38 axis facilitates uncoupling protein 1 (UCP1) induction cell-autonomously. Here, we show that ASK1 suppresses an innate immune pathway and contributes to maintenance of brown adipocytes. We report a novel chemical pull-down method for endogenous kinases using analog sensitive kinase allele (ASKA) technology and identify an ASK1 interactor in brown adipocytes, receptor-interacting serine/threonine-protein kinase 2 (RIPK2). ASK1 disrupts the RIPK2 signaling complex and inhibits the NOD-RIPK2 pathway to downregulate the production of inflammatory cytokines. As a potential biological significance, an in vitro model for intercellular regulation suggests that ASK1 facilitates the expression of UCP1 through the suppression of inflammatory cytokine production. In parallel to our previous report on the PKA-ASK1-p38 axis, our work raises the possibility of an auxiliary role of ASK1 in brown adipocyte maintenance through neutralizing the thermogenesis-suppressive effect of the NOD-RIPK2 pathway.
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Adipocitos Marrones/metabolismo , MAP Quinasa Quinasa Quinasa 5/farmacología , Proteínas Adaptadoras de Señalización NOD/efectos de los fármacos , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/efectos de los fármacos , Adipocitos Marrones/efectos de los fármacos , Adipocitos Blancos/metabolismo , Animales , Citocinas/análisis , Células HEK293 , Humanos , Inflamación/tratamiento farmacológico , Ratones , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína Desacopladora 1/efectos de los fármacosRESUMEN
Nuclear stress bodies (nSBs) are nuclear membraneless organelles formed around stress-inducible HSATIII architectural long noncoding RNAs (lncRNAs). nSBs repress splicing of hundreds of introns during thermal stress recovery, which are partly regulated by CLK1 kinase phosphorylation of temperature-dependent Ser/Arg-rich splicing factors (SRSFs). Here, we report a distinct mechanism for this splicing repression through protein sequestration by nSBs. Comprehensive identification of RNA-binding proteins revealed HSATIII association with proteins related to N6 -methyladenosine (m6 A) RNA modification. 11% of the first adenosine in the repetitive HSATIII sequence were m6 A-modified. nSBs sequester the m6 A writer complex to methylate HSATIII, leading to subsequent sequestration of the nuclear m6 A reader, YTHDC1. Sequestration of these factors from the nucleoplasm represses m6 A modification of pre-mRNAs, leading to repression of m6 A-dependent splicing during stress recovery phase. Thus, nSBs serve as a common platform for regulation of temperature-dependent splicing through dual mechanisms employing two distinct ribonucleoprotein modules with partially m6 A-modified architectural lncRNAs.
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Proteínas del Tejido Nervioso/genética , Factores de Empalme de ARN/genética , Empalme del ARN , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Núcleo Celular/genética , Células HeLa , Humanos , Proteínas del Tejido Nervioso/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Factores de Empalme de ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Factores de Empalme Serina-Arginina/genética , Factores de Empalme Serina-Arginina/metabolismo , TemperaturaRESUMEN
Delicate animal experiments and microdose clinical trials using short-lived radioisotopes require rapid preparation with high accuracy and careful attention to safety within a limited timeframe. We have developed an open-style electrowetting on dielectric (EWOD) device containing dimple structures for the rapid preparation of radiolabelled reagents. The device was demonstrated by automatic preparation of a technetium diethylenetriamine pentaacetate (99mTc-DTPA) with high chelation efficiency (99.7 ± 0.13%). Additionally, we demonstrated the single-photon emission computed tomography/computed tomography imaging of mouse kidney using the 99mTc-DTPA prepared with the EWOD device. The obtained organ tomographic images were sufficient for the evaluation of mouse models for specific diseases. These results indicate that manual radiolabelling for a small amount of nuclear medicine can be replaced by a process using the proposed EWOD device as a human error reduction technique.
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PGAM5 is a protein phosphatase located in the inner mitochondrial membrane through its transmembrane (TM) domain and is cleaved within the TM domain upon mitochondrial dysfunction. We found previously that cleaved PGAM5 is released from mitochondria, following proteasome-mediated rupture of the outer mitochondrial membrane during mitophagy, a selective form of autophagy specific to mitochondria. Here, we examined the role of cleaved PGAM5 outside mitochondria. Deletion mutants that mimic cleaved PGAM5 existed not only in the cytosol but also in the nucleus, and a fraction of cleaved PGAM5 translocated to the nucleus during mitophagy induced by the uncoupler CCCP. We identified serine/arginine-related nuclear matrix protein of 160 kDa (SRm160)/SRRM1, which contains a highly phosphorylated domain rich in arginine/serine dipeptides, called the RS domain, as a nuclear protein that interacts with PGAM5. PGAM5 dephosphorylated SRm160, and incubation of lysates from WT cells, but not of those from PGAM5-deficient cells, induced dephosphorylation of SRm160 and another RS domain-containing protein SRSF1, one of the most characterized serine/arginine-rich (SR) proteins. Moreover, phosphorylation of these proteins and other SR proteins, which are commonly reactive toward the 1H4 monoclonal antibody that detects phosphorylated SR proteins, decreased during mitophagy, largely because of PGAM5 activity. These results suggest that PGAM5 regulates phosphorylation of these nuclear proteins during mitophagy. Because SRm160 and SR proteins play critical roles in mRNA metabolism, PGAM5 may coordinate cellular responses to mitochondrial stress at least in part through post-transcriptional and pre-translational events.
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Proteínas Mitocondriales/metabolismo , Mitofagia/genética , Fosfoproteínas Fosfatasas/metabolismo , Factores de Empalme Serina-Arginina/metabolismo , Antígenos Nucleares/metabolismo , Núcleo Celular/metabolismo , Citosol/metabolismo , Células HeLa , Humanos , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Mitofagia/fisiología , Proteínas Asociadas a Matriz Nuclear/metabolismo , Fosfoproteínas Fosfatasas/genética , Fosforilación , Proteínas de Unión al ARN/metabolismo , Factores de Empalme Serina-Arginina/fisiología , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Ribosome-associated quality control (RQC) relieves stalled ribosomes and eliminates potentially toxic nascent polypeptide chains (NCs) that can cause neurodegeneration. During RQC, RQC2 modifies NCs with a C-terminal alanine and threonine (CAT) tail. CAT tailing promotes ubiquitination of NCs for proteasomal degradation, while RQC failure in budding yeast disrupts proteostasis via CAT-tailed NC aggregation. However, the CAT tail and its cytotoxicity in mammals have remained largely uncharacterized. We demonstrate that NEMF, a mammalian RQC2 homolog, modifies translation products of nonstop mRNAs, major erroneous mRNAs in mammals, with a C-terminal tail mainly composed of alanine with several other amino acids. Overproduction of nonstop mRNAs induces NC aggregation and caspase-3-dependent apoptosis and impairs neuronal morphogenesis, which are ameliorated by NEMF depletion. Moreover, we found that homopolymeric alanine tailing at least partially accounts for CAT-tail cytotoxicity. These findings explain the cytotoxicity of CAT-tailed NCs and demonstrate physiological significance of RQC on proper neuronal morphogenesis and cell survival.
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Antígenos de Neoplasias/metabolismo , Neuronas/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribosomas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Alanina/metabolismo , Línea Celular , Supervivencia Celular , Células HEK293 , Células HeLa , Humanos , Morfogénesis , Péptidos/metabolismo , Biosíntesis de Proteínas , Proteolisis , Treonina/metabolismo , UbiquitinaciónRESUMEN
Cell culturing is a basic experimental technique in cell biology and medical science. However, culturing high-quality cells with a high degree of reproducibility relies heavily on expert skills and tacit knowledge, and it is not straightforward to scale the production process due to the education bottleneck. Although many automated culture systems have been developed and a few have succeeded in mass production environments, very few robots are permissive of frequent protocol changes, which are often required in basic research environments. LabDroid is a general-purpose humanoid robot with two arms that performs experiments using the same tools as humans. Combining our newly developed AI software with LabDroid, we developed a variable scheduling system that continuously produces subcultures of cell lines without human intervention. The system periodically observes the cells on plates with a microscope, predicts the cell growth curve by processing cell images, and decides the best times for passage. We have succeeded in developing a system that maintains the cultures of two HEK293A cell plates with no human intervention for 192 h.
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Microscopía , Programas Informáticos , Animales , Línea Celular , Proliferación Celular , Humanos , Reproducibilidad de los ResultadosRESUMEN
Viral RNA in the cytoplasm of mammalian host cells is recognized by retinoic acid-inducible protein-I-like receptors (RLRs), which localize to cytoplasmic stress granules (SGs). Activated RLRs associate with the mitochondrial adaptor protein IPS-1, which activates antiviral host defense mechanisms, including type I IFN induction. It has remained unclear, however, how RLRs in SGs and IPS-1 in the mitochondrial outer membrane associate physically and engage in information transfer. In this study, we show that NUDT21, an RNA-binding protein that regulates alternative transcript polyadenylation, physically associates with IPS-1 and mediates its localization to SGs in response to transfection with polyinosinic-polycytidylic acid [poly(I:C)], a mimic of viral dsRNA. We found that despite its well-established function in the nucleus, a fraction of NUDT21 localizes to mitochondria in resting cells and becomes localized to SGs in response to poly(I:C) transfection. NUDT21 was also found to be required for efficient type I IFN induction in response to viral infection in both human HeLa cells and mouse macrophage cell line RAW264.7 cells. Our results together indicate that NUDT21 links RLRs in SGs to mitochondrial IPS-1 and thereby activates host defense responses to viral infection.